The broad, long-term aim of our research is to understand how neurotransmitters activate receptor-coupled ion channels of the Cys-loop superfamily. Focusing on the acetylcholine receptor (AChR) at the motor synapse, we address the central question of how ACh binding triggers opening of an ion channel intrinsic to the AChR. Toward understanding the binding-triggering process, we propose to (i) determine how residues at the junction of binding and pore domains link agonist binding to channel gating, (ii) delineate a comprehensive reaction mechanism underlying AChR activation, (iii) identify structures at subunit interfaces that mediate inter-subunit global coupling, and (iv) develop a water-soluble AChR ligand binding domain for studies of structure and ligand recognition. The approach combines site-directed mutagenesis, naturally-occurring mutations, single channel kinetic analysis, structural modeling, computational methods and protein biochemical and structural methods. Completion of the proposal will advance understanding of synaptic transmission and drug action at motor endplates, facilitate treatment of inherited disorders of synaptic receptors, while the general principles will provide insight into how structure gives rise to mechanism for other members of the Cys-loop receptor superfamily. PUBLIC HEALTH RELEVANCE Throughout the nervous system, moment-to-moment communication relies on post-synaptic receptors to detect nerve-released neurotransmitter and change the membrane potential. The change in membrane potential elicits a response in the receptive cell, such as muscle contraction, neuro-secretion or a change in excitability and thus altered communication with other cells. In many neurological diseases and drug addiction the interaction between neurotransmitter and post- synaptic receptor is altered, which if understood, could provide bases for rational therapy. Furthermore, many clinically useful drugs act on post-synaptic receptors, but often have unwanted side effects. Understanding both structures and molecular mechanisms of post-synaptic receptors would enable design of more specific drugs.